WHEN IN DOUBT, PIG IT OUT: A VERSATILE TRANSLATIONAL PLATFORM TO STUDY CKD, HFpEF, AND CKM SYNDROME

Abstract

Chronic kidney disease (CKD), heart failure with preserved ejection fraction (HFpEF), and the recently defined Cardiovascular-Kidney-Metabolic (CKM) syndrome are important challenges for the health care system, associated with a staggering increase in morbidity and mortality rates. Unfortunately, the complex pathophysiology of these processes remains to be fully elucidated, imposing hurdles to prevent their development and decelerate their progression.

Large animal models are critical for understanding cardio-renal pathophysiology and for the discovery of new therapies. Among them, pig models offer significant translational power to grasp cardio-renal pathophysiology and to test the feasibility and efficacy of new strategies. We recently developed and characterized swine models of CKD, HFpEF, and CKM syndrome that closely recapitulate human disease and allow for the study of the impact of biological variables, such as sex or age, on these conditions. Furthermore, data from these studies revealed key mechanisms implicated in the pathogenesis of CKD, HFpEF, and CKM syndrome and identified potential targets for intervention. This focused mini review highlights the main features of these novel swine models and discusses ongoing and future research aimed at developing novel therapies.

Chronic kidney disease (CKD) and the development of heart failure (HF).

CKD affects nearly 14% of the general population¹, 38% of US adults aged 65 years and older, and its prevalence has increased over the past two decades². The main etiologies for CKD are diabetes and hypertension, which commonly coexist with CKD and substantially magnify the risk of developing atherosclerosis³, metabolic abnormalities⁴, and cardiovascular (CV) disease^5–8. In addition, CKD is an independent CV risk factor^9–12 for the development of HF, which increases hospitalization, reduces life expectancy, results in 5-fold higher health costs compared to non-CKD patients, and imposes a huge burden on the healthcare system¹. HF affects 20–45% of men and women over 45 years of age¹³ and studies show that CKD links with longitudinal increases in left ventricular (LV) mass and volume (LV hypertrophy-LVH) and impaired LV relaxation^14,15, resulting in LV diastolic dysfunction and risk of developing HF^12,16–18 with reduced and preserved ejection fraction (HFpEF)¹⁹. Strikingly, HFpEF is present in over 50% of patients with CKD¹⁸, the rates of HFpEF in CKD increase with the loss of renal function¹⁹, and HFpEF accounts for over 50% of all hospital admissions for HF²⁰. Importantly, the scant benefits from clinical trials of HFpEF underscore its heterogeneous nature, demanding mechanistic insights into HF in CKD, their interactions, and a need for new translational models and treatments²¹ to address a major unmet need in CV research^21,22. Although these data strongly imply causation among these conditions, several aspects of causation should be considered, including strength, consistency, specificity, temporality, biological gradient, plausibility, coherence, experiments, and analogy, as insightfully reviewed by Sir Austin B. Hill²³. Furthermore, new strategies and translational interventions to slow, halt, or reverse the development and progression of HF in CKD may reduce CV deaths and support the mission of the National Institutes of Health (NIH) and the American Heart Association (AHA)^24,25.

Cardiovascular-Kidney-Metabolic (CKM) syndrome.

CKM is a new pathophysiological framework (conceptualized in early 2023 by the AHA) that describes a set of interrelated metabolic risk factors and their effects on the kidneys and CV system. CKM syndrome has four stages of progressive cardio-renal-metabolic compromise, leading to multiorgan dysfunction and a high rate of CV disease (CVD) and adverse outcomes²⁶ (Figure 1). In stage 0, patients do not exhibit any CKM risk factors (e.g., excess/dysfunctional adiposity, metabolic risk factors, CKD). In stage 1, patients exhibit excess adiposity, dysfunctional adiposity (defined as hyperglycemia or prediabetes), or both, whereas in stage 2, it is characterized by the presence of metabolic risk factors, moderate- to high-risk CKD, or both. In stage 3, patients have subclinical CVD overlapping with CKM risk factors, very high-risk CKD, or high predicted CVD risk. Finally, in stage 4, clinical CVD overlaps with CKM risk factors. This stage is divided based on the presence of CKD (defined by the Kidney Disease Improving Global Outcomes) into 4a (without kidney failure) and 4b (with kidney failure)^26,27.

Figure 1. Main features and stages of cardiovascular-kidney-metabolic (CKM) syndrome and prevalence in US adults per stage (%).

CKD: chronic kidney disease; CVD: cardiovascular disease

Recent data from the NHANES indicate that up to 90% of US adults meet the criteria for CKM stage 1 or higher²⁸. Excess adiposity, dyslipidemia, and glucose-insulin abnormalities are central to CKM syndrome and play a critical role in the pathophysiology of CKD, CVD, and CKD-to-CVD. Indeed, metabolic derangements favor a pro-oxidant and proinflammatory milieu that unfolds damaging signaling in vascular, cardiac, and renal tissues, which in turn promotes insulin resistance^26,29,30 and exerts positive feedback on inflammatory signaling, thereby building a vicious circle that facilitates target organ injury and the progression of CKM syndrome. Likewise, CKD and HF are pivotal components of CKM syndrome, being present throughout the CKM stages in more than 70% of patients²⁸. Importantly, the greatest impact of CKM syndrome on CVD morbidity and mortality is driven by the high burden of HF^26,28.

The versatility of the swine as a translational platform to study the pathophysiology of CKD, HF, and CKM syndrome.

Elucidation of mechanisms leading to HF in CKD or CKM syndrome is a significant knowledge gap²⁷. This poor understanding of cardio-renal-metabolic interactions is empowered by the lack of translational models that emulate the complexity of the human situation and allow for the identification of pathways leading to HF in CKD and CKM, which could shed light on new targets for interventions.

Animal models are critical for understanding cardio-renal pathophysiology and for the development of new therapies. A significant body of literature in the cardio-renal field originates from rodent models of HF^31,32 and models of CKD, including glomerular hypoxia^33,34 and several model of diabetes such as mice with eNOS deficiency, streptozotocin, OVE26 mice, and BTBR ob/ob mouse^35,36. For example, in a model of glomerular hypoxia achieved by knockout of HIF-1α is protective against glomerulosclerosis and glomerular type-I collagen accumulation in a mouse podocyte ablation model.

Rodents are widely used and regularly preferred over large animals as disease platforms for a variety of reasons, including lower maintenance costs and the possibility of genetic manipulations or specific interventions^37,38. However, pathophysiology in rodents is often not replicated in larger animals or humans, and differences in cardio-renal anatomy, physiology (e.g., renal and cardiac hemodynamic parameters, blood pressure, heart rate), and pathophysiology (e.g., sensitivity to develop fibrosis) in rodents compared to human kidneys hinder their potential for clinical translation (Table 1). Hence, translational platforms that could address and complement the mechanistic knowledge gained from small animal models could improve our understanding of cardio-renal-metabolic pathophysiology, facilitate the development of new treatments, and ultimately help to move the field forward.

Table 1.

Summary of main differences, similarities, advantages and disadvantages of rodent and swine systemic, cardiac, renal, and metabolic characteristics compared to humans.

Parameter	Rodent	Swine
Systemic
Anatomy and organ distribution	Similar to H	Similar to H
Organ and body size	Distant from H	Closer to H
Cardiac
Anatomy	Limited similarities to H	Similar to H
Physiological parameters	Distant from H	Closer to H
Pathophysiology	Limited similarities to H	Similar to H
Renal
Anatomy	Unipapillary	Multipapillary as in H
Physiological parameters	Distant from H	Closer to H
Pathophysiology	Often not replicated in H	Usually replicated in H
Metabolic
Metabolic rate	Distant from H	Closer to H
Genes
Genetic similarities	Distant from H	Closer to H
Inducible genetic modifications	Available and abundant	Limited
Other
Tissue availability	Low	High
Combined etiologies of CKD, HF, and/or CKM syndrome	Possible	Possible
Cost	Low	High

H: humans; CKD: chronic kidney disease; HF: heart failure; CKM: cardiovascular-kidney-metabolic. Adapted and modified^90–92 from Chade et al., Am J Physiol Renal Physiol 2018 Vol. 315 Issue 2 Pages F364-F373.

The cardiovascular and metabolic parameters in pigs are virtually identical to humans^25,39–42, allowing the use of interventions, technology, and techniques with predictable responses^25,43 highlighting the translational power of our model to grasp cardio-renal pathophysiology and to test new strategies. Furthermore, pigs express similar genetic markers compared to humans^44–46 and results from therapeutic interventions in swine were successfully replicated in clinical trials^47–52, underscoring the value of pigs as translational models. These unique advantages have been the impetus for developing swine models that recapitulate many features of CKD^25,39–41, HF^{39,44,45,53–57}, metabolic abnormalities⁵⁸ and CKM syndrome^42,59, induced by the combination of bilateral renal artery stenosis and high-cholesterol/high-carbohydrate feeding for 14 weeks (Figure 2). Induction of CKD via this approach carries minimal to no mortality compared to other swine models of CKD-HFpEF in which mortality can reach over 50%^60,61. We demonstrated that this combination exacerbates the development and progression of cardiac, renal, and metabolic abnormalities compared to the effects of each individual insult^43,62–64. These animals develop hypertension, LV remodeling and moderate fibrosis, HFpEF (a critical component that associates with adverse outcomes in CKM syndrome⁶⁵), subendocardial microvascular rarefaction, CKD⁴² (GFR around 60 mL/min), renal inflammation, cortical and medullary microvascular rarefaction, remodeling, and loss (reduced microvascular density), and fibrosis that is evident throughout the renal compartments. These cardiac and renal abnormalities were accompanied by subcutaneous and visceral adiposity, dyslipidemia, and insulin resistance⁴², the latter been consistently underscored as an important risk factor for the development of CKM⁶⁶ and HFpEF⁶⁷. More recently, we extended these studies in a new model of CKD, HF, and metabolic derangements in naturally aged swine⁴², adding age as a biological variable. This study demonstrated that some of these pathological features may be exacerbated with age, adding translational relevance to our model, as these diseases have a higher prevalence in the elderly.

Figure 2. Cardiac, renal, and metabolic phenotype in a novel swine model of CKM syndrome.

A-B) Echocardiography shows altered cardiac function and remodeling with preserved ejection fraction. C) Multi-detector computed tomography quantification of identified impaired renal hemodynamics and filtration function. D-E). Altered metabolic profile and increased adipose tissue accumulation (arrows, subcutaneous, visceral, perirenal, measured by CT). Studies were performed in normal and CKM pigs, 6–9 years old, after 14 weeks of CKM (bilateral renal artery stenosis + high-cholesterol/high-carbohydrate diet).

These traits are accompanied by a distinct renal and cardiac transcriptomic landscape. Indeed, our recent data from single-nucleus RNA sequencing (snRNA-seq) studies revealed that the kidneys of these models display a distinct set of differentially expressed genes (DEGs) predominantly in renal endothelial, proximal tubular, and connective tubular cells, which participate in inflammation and fibrosis⁶⁸. Our prior research has demonstrated that inflammation and fibrosis are key pathological features in this model^25,40–42 and are also prevalent in CKD in general, regardless of the etiology. Furthermore, comprehensive cardiac mRNA-, miRNA-, and epigenetic (MeDIP)-seq studies and their integrated analysis showed a well-defined transcriptomic profile, identifying DEGs associated with cardiac remodeling, mitochondrial function, VEGF-related signaling, and fatty acid metabolism that are pre- and post-transcriptionally regulated early in the disease^44,45,53, shedding light into pathological pathways that may foster research toward therapeutic strategies to reduce cardiovascular morbidity. Ongoing sequencing studies in the kidneys and heart of aging pigs (work in progress, unpublished) are investigating whether this transcriptomic landscape is altered or accentuated with aging, which may, in turn, pinpoint new potential targets for intervention.

Therapeutic potential

The availability of these robustly characterized models enables the development and preclinical testing of interventions targeting the major pathophysiological pathways involved in the progression of cardiac and renal disease. Below is a brief discussion of promising recently published therapeutic strategies, the pathophysiology and prior research supporting their potential, and the possibility and need for future studies to nurture their clinical translation (Figure 3).

Figure 3. Potential therapeutic targets and interventions in CKD, HFpEF, and CKM syndrome.

CKD: chronic kidney disease; HFpEF: Heart failure with preserved ejection fraction; CKM: cardiovascular-kidney-metabolic

A prominent feature of chronic kidney disease (CKD) is chronic, low-grade systemic and renal inflammation that correlates with increased cardiovascular and all-cause mortality, as demonstrated in prior studies^69–71. Our swine model develops a distinct inflammatory profile at the renal, systemic, and cardiac level, with a predominance of interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), and tumor necrosis factor (TNF)-α^39,40. Interestingly, it appears that these cytokines originate mainly from the renal system, rather than the heart, and reach the circulation, likely targeting the heart and leading to diastolic dysfunction and left ventricular remodeling³⁹. Considering that CKD is a multi-organ disease and that the pigs are fed an atherogenic diet, we cannot rule out the possibility that these inflammatory cytokines in our models might also be generated in other tissues. Furthermore, the disappointing results from clinical studies testing direct inhibition of inflammatory cytokines in cardiac failure^72–74 led to the possibility that upstream targeting of inflammatory signaling would be worth testing. We took advantage of our swine model and test a targeted intra-renal anti-inflammatory intervention via inhibition of nuclear factor kappa B (NF-κB), an upstream stimulus for the release of these cytokines, resulted in a significant decrease in renal and circulating IL-6 and TNF-α, improved renal and cardiac function, and ameliorated renal-cardiac injury^39,40, supporting the role of a renal-cardio pathophysiological axis. Future studies testing this targeted anti-inflammatory strategy in the heart, as well as investigating additional upstream modulators of inflammatory cytokines, will further explore the feasibility and potential application of modulating inflammatory signaling in cardio-renal-metabolic disorders.

Microvascular disease is a critical component in the pathophysiology of CKD and HF^1,75,76. The renal and cardiac microcirculation in the swine are very similar to humans^77,78, making it an ideal platform to assess the role of coronary microvascular dysfunction in the development of CKD, HF, and CKM syndrome. Elegant prior research in swine models^58,79,80 support the role of oxidative stress in the development of coronary microvascular dysfunction in CKD-related HFpEF, in line with our studies⁵³. Furthermore, our model displays a significant and progressive renal (cortex and medulla) and cardiac (subendocardial and subepicardial) microvascular rarefaction, remodeling, and loss^25,41,45, which is exacerbated in aging animals⁴². A loss of vascular endothelial growth factor (VEGF), a prominent mediator of vascular proliferation and repair in all vascular networks, including the kidney and the heart, appears to play a central role. Indeed, a targeted intra-renal delivery of VEGF to replenish its organ availability rescued renal MV density and renal function^41,81,82. Future studies will define whether this approach is feasible and functionally consequential in the heart of our model of HF or CKM. Although the underlying mechanisms of the loss of VEGF in the kidney or the heart are still not fully defined, our recent study postulated that epigenetic mechanisms may participate in the loss of VEGF and/or altered VEGF signaling (e.g., altered VEGF-related genes)^45,83. These findings open the possibility of considering the use of epigenetic modulators (e.g., TET enzyme inhibitors, vitamin C) as a potential renal and cardioprotective intervention, which could be tested in future studies.

Mitochondria are the primary source of cellular energy and a critical modulator of cellular processes, including oxidative phosphorylation, calcium homeostasis, cell proliferation, and programmed cell death. A healthy mitochondrion is crucial for the contraction and relaxation of the heart, and mitochondrial dysfunction has been reported in various cardiovascular and metabolic diseases. The swine model of CKD and HF presents a distinct set of several mitochondria-related genes dysregulated at pre- and post-transcriptional levels that are associated with cardiac dysfunction, injury, and remodeling^44,53. These studies were performed in young animals and was interesting to observe that cardiac dysfunction and remodeling were accompanied by altered mitochondrial morphology (electron microscopy^44,53), increased generation of reactive oxygen species (H₂O₂) in the heart, upregulated expression of mitochondria-related genes primarily implicated in reactive oxygen species production and antioxidant defenses, and downregulated expression of genes implicated in ATP production and respiration but with a relatively preserved cardiac mitochondrial function (Oroboros)⁵³. It is possible that the age of the animals may have contribute to their resilient phenotype and that the disparity between cardiac mitochondrial morphology, function, and expression of antioxidant genes suggest active compensatory (e.g., preserved mitochondrial respiration) but insufficient (e.g., altered morphology, persistence of diastolic dysfunction and remodeling) mechanisms that could be abated as the disease progresses. Similarly, this resilient phenotype may be reflected in the responses to therapeutic interventions, demanding caution when considering a potential clinical translation. For example, patients with CKD, HFpEF, or CKM syndrome usually present multiple comorbidities and advanced age, thus a poorer response to therapy compared to animal models is possible. Nevertheless, the findings of our study⁵³ may set the stage for testing potential pre- and post-transcriptional modulation of mitochondrial-related genes (e.g., microRNA inhibition) and pave the way for extending these studies in the novel swine model of aging combined with cardio-renal-metabolic abnormalities in future studies.

Preclinical testing of cell-based therapies has been previously conducted in several swine models of renal and cardiovascular disease. For example, intra-renal delivery of mesenchymal stem cells (MSCs) or their daughter extracellular vesicles, which exert important pro-angiogenic and immunomodulatory effects, alone or in combination with renal revascularization, preserved the structure and function of swine stenotic kidneys^50,84,85. Importantly, these benefits were extended to the hearts of pigs with renovascular hypertension, underscoring the importance of reno-protective strategies to preserve cardiac structure and function. Notably, MSC therapy has been translated into clinical trials in patients with renovascular disease^47,51, showing an excellent safety profile and unique potential to ameliorate renal hypoxia and improve kidney function when administered alone or in conjunction with renal artery stenting. However, whether targeted intra-coronary or intra-renal MSC delivery protects the hearts and kidneys of pigs with CKD, HFpEF, or CKM syndrome remains unknown and warrants further investigation.

Lastly, strategies aimed at modulating the cardiac and renal expression of micro-RNAs, such as systemic, intra-renal, or intra-coronary delivery of miRNA inhibitors (antagomiRs), which have shown beneficial effects in different models of HF^86–88 and renal disease⁸⁹, remain to be tested in pigs with CKD, HFpEF, or CKM syndrome. Future studies focused on modulating the expression of miRNAs and their primary targets identified to participate in the pathogenesis of these conditions (e.g., modulation of inflammatory or pro-fibrotic signaling, mechanisms of vascular repair), could yield significant benefits and potentially improve the cardiovascular outcomes of patients with CKD, HFpEF, or CKM syndrome.

Conclusions and Perspectives

The complexity of CKD, HFpEF, and the recently defined CKM syndrome necessitates the development of suitable models to study their pathophysiology, thereby facilitating the discovery of novel therapeutic strategies. The similarities of renal and cardiac anatomy and physiology between swine and humans offer a unique preclinical platform with strong translational power. Our recently developed swine models of CKD, HFpEF, and CKM syndrome reveal important mechanisms involved in the development and progression of these processes and demonstrate their potential for reversibility via targeted interventions (e.g., anti-inflammatory strategies, therapeutic angiogenesis). Ongoing efforts and future studies focused on additional mechanisms underlying renal and cardiac injury, as well as the impact of biological variables (e.g., sex, aging) on the development of these diseases and responses to treatments, may lead to new tailored strategies to treat and prevent these conditions of growing prevalence. Our prior research included males and females and we did not observe differences in the cardiac or renal disease phenotype as well as in their responses to treatments^{39,44,45,53,81}. Yet, the animals in these studies were juvenile, and swine’s sexual maturation is influenced by age, weight, and the presence of the male boar, warranting the need of additional studies considering these factors. Working with aging large animal models carries inherent challenges related to their high cost, size, and possible age-related differences in their sensitivity to insults and responses to therapies. In addition, suppliers typically do not have enough aged swine for widespread studies, limiting the practical use of aged swine and warranting efforts to improve resources for aged swine. Despite these obstacles, we recently demonstrated that aging pigs (6–9 years old) can fully recapitulate the phenotype of CKD, HFpEF, and CKM syndrome⁴², opening a promising avenue for further translational research in the field.